EP0245138B1 - Mikrobiologisches Verfahren zur Gewinnung eines Proteins durch Züchtung eines mutanten Bakterienstammes - Google Patents

Mikrobiologisches Verfahren zur Gewinnung eines Proteins durch Züchtung eines mutanten Bakterienstammes Download PDF

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EP0245138B1
EP0245138B1 EP87400813A EP87400813A EP0245138B1 EP 0245138 B1 EP0245138 B1 EP 0245138B1 EP 87400813 A EP87400813 A EP 87400813A EP 87400813 A EP87400813 A EP 87400813A EP 0245138 B1 EP0245138 B1 EP 0245138B1
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expression
protein
strain
process according
microbiological process
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EP0245138A1 (de
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Richard Legoux
Pascal Leplatois
Evelyne Liauzun Joseph
Brigitte Niaudet
Willem Roskam
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Elf Sanofi SA
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Elf Sanofi SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor

Definitions

  • the subject of the present invention is a microbiological process making it possible to obtain a protein by culture of gram-negative bacteria carrying a mutation of a particular type, into which a DNA sequence coding for the precursor of said protein has been introduced. .
  • the invention finds its application in numerous technical fields and more particularly in the preparation of medicaments in which the active principle is a protein.
  • proteins acquire their biological activity only after being subjected to post-translational maturation.
  • proteins are biosynthesized in the cell cytoplasm in the form of a precursor, then exported out of the cytoplasm, the first step of this maturation is often the elimination by an enzymatic route of a sequence of the N-terminal end, called signal peptide, of this precursor.
  • signal peptide a sequence of the N-terminal end
  • beta-lactamase TEM-1 alkaline phosphatase, both produced for example by the species Escherichia coli
  • protein A produced by the species Staphylococcus aureus
  • the alpha-amylase produced for example by the species Bacillus amyloliquefaciens
  • proteins of prokaryotic origin as well as human growth hormone, human insulin, tissue activator of plasminogen and epidermal growth factor, proteins produced by eukaryotic cells.
  • gram-negative bacteria can be used, previously transformed with a plasmid carrying a DNA sequence coding for the natural precursor of a protein, with a view to obtaining said protein (Gray GL and al., Biotechnology 2 (1984) 161-165; Gray GL et al., Gene 39 (1985) 247-254).
  • the cellular machinery ensures in the cytoplasmic compartment the transcription of the DNA sequence and then the translation of the corresponding messenger RNA; the precursor thus biosynthesized then undergoes during or after the crossing of the cytoplasmic membrane a cut or a degradation enzymes eliminating its signal peptide; the protein, thus corresponding to the precursor devoid of its signal peptide, accumulates in the periplasm of the bacterium from which it can be collected.
  • DNA sequence codes for a precursor different from the natural precursor (that is to say as it is synthesized in the cells which usually produce it) as regards the signal peptide;
  • a precursor corresponding to a protein of eukaryotic origin associated with the signal peptide of a protein of bacterial origin has, for example, been synthesized (Talmadge K. et al., Proc. Natl. Acad. Sci. USA, 77 ( 1980) 3988-3992; Gray GL et al., Gene 39 (1985) 247-254).
  • bacteria can be the object of mutations limiting, even suppressing, the expression of operons whose transcription can only be initiated after fixing, at the level of the regulatory elements of said operons, of the AMPc- complex.
  • CRP resulting from the association of cyclic adenosine acid 3 ⁇ , 5 ⁇ -phosphoric (or cAMP) and the CRP protein (for Cyclic AMP Receptor Protein) also called CAP protein (for Catabolite Activator Protein).
  • CRP resulting from the association of cyclic adenosine acid 3 ⁇ , 5 ⁇ -phosphoric (or cAMP) and the CRP protein (for Cyclic AMP Receptor Protein) also called CAP protein (for Catabolite Activator Protein).
  • the Applicant has specifically chosen another research path by focusing on the bacterial host.
  • gram-negative bacteria whose chromosome carries at least one mutation limiting, even suppressing, the expression of operons whose transcription depends on the fixation of the cAMP- complex CRP, were the hosts of choice for the industrial production of biosynthesized proteins in the form of a precursor.
  • the present invention therefore relates, according to a first aspect, to a microbiological process for obtaining a protein, by culture of gram-negative bacteria, into which has been introduced a DNA sequence coding for a precursor of this protein, characterized in that one implements a strain of bacteria whose chromosome carries at least one mutation limiting, even suppressing, the expression of the operons for which the transcription depends on the fixation of the AMPc-CRP complex.
  • the mutations concerned are those which tend to reduce the intracellular concentration of cAMP, those which limit or suppress the synthesis of the CRP protein in a functional form, those which lead to the synthesis of the CRP protein in a mutated form and of which it results in the formation of cAMP-CRP complexes such that their attachment to DNA is prevented or is made ineffective, as well as those which modify the binding site on DNA of the cAMP-CRP complex, so that the attachment of said complex is prevented or made ineffective.
  • They can be conditional mutations whose expression depends on a parameter such as, for example, temperature.
  • mutations those that affect the chromosomal gene cya itself or a DNA sequence regulating its expression, such that adenylate cyclase, for which this gene codes, is not synthesized or is synthesized in a form such that it is unable to exercise its specific activity - namely the transformation of adenosine triphosphate (or ATP) into cAMP - are well characterized.
  • mutations which affect the chromosomal gene crp itself or a DNA sequence regulating its expression such that the CRP protein, for which this gene codes, is not synthesized or is synthesized under a shape mutated such that there cannot be formation of AMPc-CRP complexes sufficiently stable to allow their efficient attachment to DNA.
  • These mutations will be, for convenience, hereinafter designated cya mutation and crp mutation.
  • Cya and crp mutations can be of all kinds: they can be in particular a substitution mutation, an insertion mutation, an inversion mutation or even for example a deletion mutation.
  • the mutation may affect only one pair of nucleotides, but since such a point mutation is reversible, a mutation which, simultaneously affecting several pairs of nucleotides, is irreversible, is preferred. It can also be multiple point mutations.
  • the cAMP is not synthesized and there can be no formation of the cAMP-CRP complex; the addition of exogenous cAMP makes it possible to overcome this defect in synthesis and restores the disturbed cellular functions.
  • the CRP protein is no longer synthesized in a functional form and, likewise, there cannot be formation of the cAMP-CRP complex; the addition of exogenous cAMP is here powerless to restore the disturbed cellular functions.
  • the invention advantageously relates to the microbiological process defined above, in which a strain of bacteria is used, the chromosome of which carries at least one mutation affecting the group formed by the cya gene and the DNA sequences regulating its expression, the set formed by the crp gene and the DNA sequences regulating its expression, or both.
  • the bacterial strains used according to the invention can belong to any gram-negative species in which the cAMP, coupled to the CRP protein, controls the expression of various operons.
  • the use of such bacterial strains for industrial production is all the more advantageous since they exhibit remarkable resistance to the action of numerous bacteriophages (Sushil Kumar, J. Bact., 125 (1976) 545-555).
  • Escherichia coli is a species of choice.
  • the invention relates in another aspect to said microbiological process characterized in that the mutant bacterial strain used is a strain of the species Escherichia coli .
  • the Applicant has more particularly selected two mutant strains of Eschericha coli deposited on February 17, 1986, from the National Collection of Cultures of Microorganisms (CNCM, Paris, France).
  • One strain carries a point crp mutation (deposit No. I-528); the other strain carries a cya mutation by deletion nonpoint (deposit No. I-529).
  • the invention preferably relates to said microbiological process characterized in that this mutant strain characteristics of either of the strains deposited at the CNCM under the No. I-528 and I-529.
  • the process of the invention allows numerous proteins to be obtained. It should be understood that it does not matter whether the protein, which it is desired to obtain, is naturally biosynthesized or not in the form of a precursor.
  • the method may also be suitable for obtaining an unnatural protein; it may for example be a hybrid protein, the primary structure of which has been chosen from two or more known proteins. In all cases, it suffices for a DNA sequence coding for said protein associated at its N-terminal end with a peptide playing the role of a signal peptide to be prepared.
  • the method according to the invention is particularly advantageous for obtaining human growth hormone (hereinafter designated in the text by the symbol hGH).
  • FIG. 1 represents the DNA sequence coding for one of the natural precursors of hGH as it is isolated from the cells of the pituitary gland where it is synthesized, as well as the amino acid sequence of said precursor: the first 26 amino acids (counted in the figure starting from the amino acid in position - 26) constitute the signal peptide and the other 191 amino acids (here numbered from 1 to 191) are those of hGH proper ( hereinafter called in the text and examples "mature hGH").
  • the letters A, C, T and G have the usual meaning and denote the bases respectively: adenine, cytosine, thymine and guanine.
  • the mutant bacteria require that the mutant bacteria contain a DNA sequence coding for the precursor of the protein of interest.
  • precursor is meant the natural precursor of the protein when it exists, any derivative thereof obtained by modification made to its signal peptide, as well as any precursor which associates with said protein a peptide capable of playing the role of a signal peptide and which can in particular be chosen from known prokaryotic and eukaryotic signal peptides or from their derivatives.
  • the DNA sequence coding for the precursor of said protein can be introduced into the bacterium, carried by an expression vector capable of replication, such as a plasmid. It is clear that the regulatory elements (promoter in particular) necessary for the expression of said sequence must be placed in its vicinity in such a way that they can play their role.
  • the plasmids which can be used are for example those described in the prior art (Gray G. L. et al., Gene 39 (1985) 247-254) provided that they are capable of replicating in the bacterial strain used. It was verified, for the choice of these plasmids, that it was not necessary that, in the construction used, the DNA sequence coding for the precursor of the protein of interest is inducible. It is also important that the said sequence is present in several copies.
  • the Applicant has precisely constructed and selected several plasmids which may be present, after replication in bacterial cells, at a rate of 50 to 300 copies per cell, each copy carrying a copy of the DNA sequence coding for the protein precursor. ; on February 17, 1986, it deposited with the CNCM the E. coli MC 1061 strain transformed by one of these plasmids; This strain has the deposit No. I-530. For convenience, reference will be made in the following description in the plasmid deposited at the CNCM under No. I-530.
  • This plasmid also designated below by its internal reference p163.1, carries a DNA sequence coding for one of the natural precursors of hGH. This sequence, represented in FIG.
  • RNA polymerase recognition site site centered on its nucleotide placed in position - 35 and as defined by Takanami M. et al.
  • Figure No. 2 shows, for said plasmid a functional map showing the location and orientation of the main DNA sequences which are denoted symbolically as follows:
  • the invention relates, according to another aspect, said microbiological method characterized in that the DNA sequence coding for the precursor of the protein of interest, accompanied by regulatory elements allowing its expression, is carried by a plasmid; it relates in particular to said microbiological process characterized in that said DNA sequence is carried by a plasmid having the general characteristics of the plasmid deposited at the CNCM under the No. I-530.
  • the invention relates, according to another aspect, said microbiological method characterized in that said bacterial strain contains a DNA sequence coding for the precursor of hGH.
  • the invention relates in a further aspect the bacterial strains deposited with the CNCM under the No. I-528 and I-529.
  • the invention further relates in another aspect the plasmid deposited at the CNCM under the No. I-530.
  • plasmid deposited at the CNCM under No. I-530 three other plasmids are mentioned in the examples: these plasmids designated by the internal references p164,1, p200,3 and p212,6.
  • Plasmid p164.1 is very close to plasmid p163.1; it differs essentially from it by the absence of the UV5 mutation.
  • Plasmid p212.6 is close to plasmid p163.1.
  • Figure No. 3 gives a functional map showing the location and orientation of the main DNA sequences which are denoted symbolically as follows:
  • Plasmid p200.3 is a plasmid equivalent to plasmid p212.6 chosen for its non-repressible nature for the expression of the DNA sequence coding for the precursor of hGH.
  • the DNA sequence encoding one of the natural precursors of hGH scope by the plasmids and p212,6 p200,3 differs from the sequence shown in Figure No. 1 with two substitutions: the ACC codon was substituted codon ACA coding for the amino acid placed in position - 24 and similarly the codon TCT has been substituted for the codon TCC coding for the amino acid placed in position - 22.
  • the tests carried out consisted in cultivating the host-vector pairs concerned previously prepared (cf. ⁇ 2.1) under conditions such that the host is able to express the DNA sequence coding for the precursor of hGH (cf. ⁇ 2.2) the expression being if necessary induced (cf. ⁇ 2.3), collecting the proteins contained in the periplasmic space (cf. ⁇ 2.4) and assaying the periplasmic hGH (cf. ⁇ 2.5).
  • An isolated colony obtained on solid medium (LB medium + agar) is suspended in 5 ml of medium (LB medium), having the following characteristics, supplemented with ampicillin at a rate of 100 ⁇ g / ml : - basic components introduced before autoclaving Pancreatic casein hydrolyzate (DIFCO Bactotryptone) 10 g Yeast extract 5 g Sodium chloride 5 g Distilled water qsq 1 l - pH adjusted to 7.3 before autoclaving
  • DIFCO Bactotryptone Pancreatic casein hydrolyzate
  • the bacterial suspension is diluted in LB medium so as to obtain an Optical Density value read at 600 nm - OD 600 nm - (spectronic spectrophotometer spectronic 20) Bausch-Lomb) close to 0.05.
  • IPTG isopropyl- ⁇ -D-thiogalactose
  • a sample of the suspension as obtained in 2.2.d. (case where induction is not required) or as obtained after a determined induction time, is removed and treated (centrifugation for 5 min at 6000 g, elimination of the supernatant and resuspension of the bacteria in the LB medium ) so that it has an OD 600 nm close to 10.
  • the suspension obtained in 2.4.a is centrifuged for 5 min at 6000 g.
  • the suspension obtained in 2.4.b. is centrifuged for 5 min at 6000 g.
  • the pellet is gently taken up at constant volume in a solution B corresponding to solution A to which sucrose is added at the rate of 15 g per 100 ml and prepared immediately.
  • the suspension is left for 10 min at 20 ° C.
  • the centrifuge tubes are placed in melting ice.
  • the suspension thus prepared (whose OD at 600 nm is close to 10) is left for 5 min at 0 ° C.
  • the suspension obtained in 2.4.c. is centrifuged for 10 min at 18,000 g.
  • the supernatant is collected and contains proteins with a periplasmic location.
  • the supernatant obtained in 2.4.d. is subjected to a radioimmunoassay of human growth hormone (kit125 I-hGH COATRIA ® - Biomérieux, France).
  • the proportion of hGH labeled with iodine 125 is inversely proportional to the amount of hGH in the sample to be assayed.
  • the periplasmic hGH was present for approximately 98% of the molecules in its mature form, whatever the bacterial strain and the plasmid used.
  • mutant strain (strain CNCM I-528) is capable of producing periplasmic hGH with greater efficiency than the non-mutant strain (strain T), the production being more than doubled.
  • mutant strain (strain CNCM I-528) is capable of producing periplasmic hGH in a significantly increased amount (increase of approximately 30% in one of the cases and increase of approximately 70% in the other case) and this whether or not the DNA sequence coding for the precursor of hGH is, for its expression, under the control of signals pondered to be sensitive to the combined action of cAMP and of the protein CRP .
  • mutant strain (strain CNCM I-528) is capable of producing periplasmic hGH with greater efficiency than the non-mutant strain (strain T), the production being more than doubled.
  • mutant strain CNCM I-529 has the capacity to produce periplasmic hGH with greater efficiency than a parental strain from which it differs, under the chosen operating conditions, only by the expression of a cya mutation.
  • strain A whose chromosome carries a cya mutation by deletion and a crp mutation by deletion and the strain I-529 whose chromosome carries the same cya mutation by deletion are capable of identical performances.
  • Example E further emphasizes the very first-rate advantage provided by the use of such bacteria: the increase observed is not only absolute but also relative, the mutant strains restricting the production of their own localized proteins. normally periplasmic, which makes it easier to purify the target protein from the contents of the periplasm.

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Claims (13)

  1. Mikrobiologisches Verfahren zur Herstellung eines Proteins durch Kultivierung Gramnegativer Bakterien, in die eine DNS-Sequenz eingeführt wurde, die für einen Vorläufer dieses Proteins codiert, dadurch gekennzeichnet, daß ein Bakterienstamm eingesetzt wird, dessen Chromosom mindestens eine Mutation aufweist, die dazu führt, die Fixierung des cAMP-CRP-Komplexes (d.h. des Komplexes von cyclischem Adenosinmonophosphat, cAMP, mit dem Rezeptorprotein für cyclisches AMP, CRP) zu verhindern oder wenig wirksam zu machen, so daß die Expression der Operons, bei denen die Transcription durch die Fixierung des cAMP-CRP-Komplexes kontrolliert wird, unterdrückt oder begrenzt wird.
  2. Mikrobiologisches Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß das Chromosom der betreffenden Bakterien mindestens eine Mutation aufweist, welche die durch das Gen cya und die seine Expression regulierenden DNS-Sequenzen gebildete Einheit beeinträchtigt.
  3. Mikrobiologisches Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß das Chromosom der betreffenden Bakterien mindestens eine Mutation aufweist, welche die durch das Gen crp und die seine Expression regulierenden DNS-Sequenzen gebildete Einheit beeinträchtigt.
  4. Mikrobiologisches Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß das Chromosom dieser Bakterien mindestens eine Mutation, welche die durch das Gen cya und die seine Expression regulierenden DNS-Sequenzen gebildete Einheit beeinträchtigt, sowie mindestens eine Mutation aufweist, welche die durch das Gen crp und die seine Expression regulierenden DNS-Sequenzen gebildete Einheit beeinträchtigt.
  5. Mikrobiologisches Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, daß die eingesetzte Bakterienstammutante ein Stamm der Art Escherichia coli ist.
  6. Mikrobiologisches Verfahren nach den Ansprüchen 2 und 5, dadurch gekennzeichnet, daß die Bakterienstammmutante die Eigenschaften des unter der Nr. I-529 bei der Hinterlegungsstelle CNCM hinterlegten Stamms aufweist.
  7. Mikrobiologisches Verfahren nach den Ansprüchen 3 und 5, dadurch gekennzeichnet, daß die Bakterienstammmutante die Eigenschaften des unter der Nr. I-528 bei der Hinterlegungsstelle CNCM hinterlegten Stamms aufweist.
  8. Mikrobiologisches Verfahren nach den Ansprüchen 4 und 5, dadurch gekennzeichnet, daß die Bakterienstammmutante ein Stamm von Escherichia coli ist, dessen Chromosom zwei Deletionen aufweist, von denen die eine die durch das Gen cya und die seine Expression regulierenden DNS-Sequenzen gebildete Einheit und die andere die durch das Gen crp und die seine Expression regulierenden DNS-Sequenzen gebildete Einheit beeinträchtigt.
  9. Mikrobiologisches Verfahren nach einem der Ansprüche 1 bis 8, dadurch gekennzeichnet, daß die für den Vorläufer des interessierenden Proteins codierende DNS-Sequenz, zusammen mit Regulierungselementen, die seine Expression erlauben, in einem Plasmid enthalten ist.
  10. Mikrobiologisches Verfahren nach einem der Ansprüche 1 bis 9, dadurch gekennzeichnet, daß der Bakterienstamm eine DNS-Sequenz enthält, die für einen Vorläufer des menschlichen Wachstumshormons codiert.
  11. Mikrobiologisches Verfahren nach Anspruch 10, dadurch gekennzeichnet, daß die betreffende DNS-Sequenz in einem Plasmid enthalten ist, das die allgemeinen Charakteristika des unter der Nr. I-530 bei der Hinterlegungsstelle CNCM hinterlegten Plasmids aufweist.
  12. Bakterienstamm, der unter der Nr. I-528 bei der Hinterlegungsstelle CNCM hinterlegt ist.
  13. Bakterienstamm, der unter der Nr. I-529 bei der Hinterlegungsstelle CNCM hinterlegt ist.
EP87400813A 1986-04-10 1987-04-10 Mikrobiologisches Verfahren zur Gewinnung eines Proteins durch Züchtung eines mutanten Bakterienstammes Expired - Lifetime EP0245138B1 (de)

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Application Number Priority Date Filing Date Title
AT87400813T ATE92962T1 (de) 1986-04-10 1987-04-10 Mikrobiologisches verfahren zur gewinnung eines proteins durch zuechtung eines mutanten bakterienstammes.
LV960299A LV5811A4 (lv) 1986-04-10 1996-07-22 Panemiens olbaltumvielas iegusanai

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FR8605133A FR2597114B1 (fr) 1986-04-10 1986-04-10 Procede microbiologique pour l'obtention d'une proteine par culture d'une souche bacterienne mutante
FR8605133 1986-04-10

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EP0245138A1 EP0245138A1 (de) 1987-11-11
EP0245138B1 true EP0245138B1 (de) 1993-08-11

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US (1) US4945047A (de)
EP (1) EP0245138B1 (de)
JP (4) JP2528872B2 (de)
CA (1) CA1304021C (de)
DE (1) DE3786947T2 (de)
ES (1) ES2059401T3 (de)
FR (1) FR2597114B1 (de)

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US5284768A (en) * 1988-08-24 1994-02-08 Sanofi Signal peptide, DNA sequences coding for the latter, expression vectors carrying one of these sequences, gram-negative bacteria transformed by these vectors, and process for the periplasmic production of a polypeptide
FR2636644B1 (fr) * 1988-08-24 1990-12-28 Sanofi Sa Sequence d'adn participant a la regulation de l'expression d'une sequence d'adn codant pour un precurseur d'un polypeptide, vecteurs d'expression et procede de production periplasmique du polypeptide
US5279947A (en) * 1988-08-24 1994-01-18 Sanofi DNA sequence participating in the regulation of the expression of a DNA sequence coding for a precursor of a polypeptide, expression vectors and process for the periplasmic production of the polypeptide
WO2013068445A1 (en) 2011-11-09 2013-05-16 Sanofi Diacylglycerol lipase and uses thereof

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JPS59125895A (ja) * 1983-01-07 1984-07-20 Rikagaku Kenkyusho ヒト生長ホルモン遺伝子の合成法
US4755465A (en) * 1983-04-25 1988-07-05 Genentech, Inc. Secretion of correctly processed human growth hormone in E. coli and Pseudomonas
JPS60221091A (ja) * 1983-12-21 1985-11-05 Kyowa Hakko Kogyo Co Ltd 新規プロモ−タ−
JPS60234584A (ja) * 1984-05-09 1985-11-21 Morio Ikehara 組換プラスミド

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FR2597114B1 (fr) 1994-02-25
DE3786947T2 (de) 1994-02-24
JP2708403B2 (ja) 1998-02-04
JPH08228789A (ja) 1996-09-10
DE3786947D1 (de) 1993-09-16
EP0245138A1 (de) 1987-11-11
FR2597114A1 (fr) 1987-10-16
CA1304021C (en) 1992-06-23
US4945047A (en) 1990-07-31
JPH09224685A (ja) 1997-09-02
JP2661894B2 (ja) 1997-10-08
JP2528872B2 (ja) 1996-08-28
ES2059401T3 (es) 1994-11-16
JPS637793A (ja) 1988-01-13

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